A cell-free protein synthesis system using a cell extract has been mainly utilized for identification of gene products and for investigation of their functions. For example, the system enables to analyze synthesized protein functions in the enzymatic activity and DNA binding capability and the like, or determine the molecular weight of translated products by labeling them with radioisotopes. Recently, techniques for increasing the amount production drastically in the system have been developed (e.g. see Patent References 1 and 2), and the system has been utilized also for protein structure analysis by X-ray crystallography, NMR and the like.
As extracts for carrying out the translation reaction, those derived from E. coli, wheat germ, and rabbit reticulocyte are commercially available. In an E. coli extract, it is known that transcription-translation coupled reaction can be used for synthesizing a protein directly from DNA. Such a method as using an E. coli S30 extract has been systematically developed by Zubay et al (e.g. see Non-patent Reference 1). The S30 extract contains ribosomes that are necessary for translation of mRNA; aminoacyl-tRNA synthetase; and initiation factors (IF), elongation factors (EF) and release factors (RF) for polypeptide chain synthesis. When a DNA template is used for protein synthesis, a DNA construct, in which a target protein gene is inserted downstream of a strong promoter (typically T7 promoter), is added in the system together with a T7 RNA polymerase and ribonucleotides to couple both reactions of transcription and translation. Due to the requirement of ATP and GTP for synthesis of aminoacyl-tRNA and translation reaction with mRNA, these are added to the cell-free system as energy sources with regeneration systems such as a creatine kinase/creatine phosphate system. With the above components, protein synthetic reaction in a cell is reconstituted in vitro.
Such an E. coli S30 extract can be prepared by growing E. coli in an appropriate culture medium, homogenizing the obtained cells, and centrifuging the cell homogenate at 30,000×g to obtain the prescribed supernatant. Generally, the culture temperature of E. coli is 37° C., and the cells are harvested at the growth stage from middle to late logarithmic growth phase of an elevated protein synthetic activity (e.g. see Patent Reference 3). The reason for use of the cells in logarithmic growth phase is that the synthetic activity of S30 extract is notably decreased when the growth stage of E. coli enters into stationary phase from logarithmic growth phase.
However, as the cell number of E. coli in a unit culture medium in the logarithmic growth phase is fewer than that in the stationary phase, there is a problem that production yield of extracts prepared from cells in the logarithmic growth phase is low. In addition, the growth time of E. coli is about 20 minutes in optimum growth conditions and this rapid growth causes another problem that the protein synthetic activity of S30 extract decreases due to the overgrowth of the cells if the cell harvesting time is not proper but late.
Recently, a transcription-translation coupled reaction system using eukaryotic cells such as wheat germ, rabbit reticulocyte or the like has been developed, however, it is necessary for such a system to use eukaryotic cells only at the specific stage of differentiation such as an early stage of development. In the conventional cell extracts, it is also a problem to cause an inactivation of transcription or translation system by degradations of mRNA and ribosomes affected by a group of contaminated nucleases, translation-inhibitory protein factors, proteases or the like during producing extracts or protein synthetic reaction.
In addition, in the recent post-genomic research field, the comprehensive analysis of structures and functions of various kinds of proteins has started, thus, it has become an essential object to improve the productivity and the workability of protein synthesis.
[Patent Reference 1]
Japanese Patent Kokoku publication No. JP-B-H07-110236.
[Patent Reference 2]
Japanese Patent Kokai publication No. JP-A-H04-200390
[Patent Reference 3]
Gazzete of International Publication No. 2001/83805
[Non-Patent Reference 1]
Geoffrey Zubay, Annual Review of Genetics, 1973, Vol. 7, pp. 267-287